CODE No.:B1D111

DIAPHORASE I (Di-1)

[EC 1. 6. 99. -]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 1,000 U/mg protein
Contaminants	:	(as Diaphorase activity = 100%)
		Adenylate kinase	<0.01%
		NADH oxidase	<0.01%

PROPERTIES

Molecular weight	:	ca. 30,000
Optimum pH	:	8.0	(Fig.1)
pH stability	:	7.5 - 9.5	(Fig.2)
Isoelectric point	:	4.7
Optimum temperature	:	70°C
Thermal stability	:	No detectable decrease in activity up to 50°C.	(Fig.3, 4)
Michaelis constants	:	See Table 1
Substrate specificity	:	See Table 1
Effectors	:	cations and anions	(Fig.5,6)

STORAGE

stable at -20 to 5°C for at least one year

APPLICATION

The enzyme is useful for the measurement of various dehydrogenase reactions in visible spectral range.

ASSAY

Principle

The change in absorbance is measured at 600 nm according to the following reaction.

Unit Definition

One unit of activity is defined as the amount of Di-1 that reduces 1 μmol of DCIP per minute at 30°C.

Solutions

1. Buffer solution ; 500mM Tris-HCl, pH8.5
2. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O/10mL distilled water)
3. 2,6-Dichlorophenolindophenol (DCIP) solution ; 1.2 mM (2.0 mg DCIP sodium salt·2H₂O/5mL distilled water) (prepare freshly)

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 1.0 to 2.0 U/mL with 50mM potassium phosphate buffer, pH 7.5.

Procedure

1. Prepare the following reaction mixture and pipette 2.85 mL of reaction mixture into a cuvette.

   Solution I	3.00mL
   Solution II	2.28 mL
   H2O	23.22 mL

2. Incubate at 30°C for about 3 minutes.
3. Add 0.15 mL of Solution III and 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 600 nm per minute (ΔAbs(test)) in linear portion of curve. Repeat the Procedure 3 using distilled water in place of enzyme solution, and ΔAbs(blank) is obtained.

Calculation

• d.f. ; dilution factor
• 19 ; millimolar extinction coefficient of DCIP (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Mains, I., Power, D.M., and Thomas, E.W. ; Biochem. J., 191, 457 (1980)

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Table 1. SUBSTRATE SPECIFICITY OF DIAPHORASE I
Acceptor	DCIP*1		NBT*2		INT*3		FMN*4
Km Acceptor (mM)	0.015		0.15		0.40		-
Km NADH (mM)	0.50		0.02		0.07		-
Km NADPH (mM)	0.52		0.19		0.50		-
Optimum pH	0.8		>10		7.5		<6.5
Activity NADH (U/mg)	1,200		225		290		18
Activity NADPH (U/mg)	4		150		120		-
Assay Mixture	Tris-HCl		TEA		Phosphate		Phosphate
	(pH 8.5)	50mM	(pH 7)	50mM	(pH 7.5)	96mM	(pH 7)	88mM
	NAD(P)H	1mM	NAD(P)H	1mM	NAD(P)H	1mM	NADH	0.2mM
	DCIP	0.06mM	NBT	0.5mM	INT	3mM	FMN	0.13mM
			Triton X-100	0.1%	(2 %-DMSO)
					BSA*5	1mg/mL
Wavelength for measurement (nm)	600		550		492		340
Extinction coefficient (cm2/μmol)	19		12.4		19.2		6.2

REFERENCE

• *1 2,6-Dichlorophenolindophenol
• *2 Nitro blue tetrazolium
• *3 p-Iodonitrotetrazolium violet
• *4 Flavin mononucleotide
• *5 Bovine serum albumin

Effect of BSA on the activity of DIAPHORASE I:

BSA stimulates the activity with INT as electron acceptor and the activation can be increased 30 fold with concentrations above 1 mg/mL BSA (Fig. 10). The extent of activation for DCIP is about 35 %, whereas the activities with NBT and FMN are not affected by BSA.

Effect of Triton X-100 on the activity of DIAPHORASE I:

The activity with NBT is little in the absence of Triton X-100, but is greatly increased by the addition of Triton X-100 (Fig. 8). On the other hand, Triton X-100 has no effect on the activities with DCIP, INT and FMN.

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